Piezoelectric ceramic sensor and sensor array for detection of molecular makers

ABSTRACT

A sensor is provided for the detection of a marker in a sample in which the sensor includes a high frequency 500 kHz-1 GHz piezoelectric ceramic resonator, with the system measuring resonant frequency change. In one embodiment, the piezoelectric sensor operates in the thickness extensional (TE) mode, with the high frequency and TE mode permitting fabrication of an exceptionally small size sensor capable of being arrayed in a handheld unit.

RELATED APPLICATIONS

This Application claims rights under 35 USC §119(e) from U.S. Application Ser. No. 61/103,268 filed Nov. 20, 2007, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to a high-frequency ceramic piezoelectric sensor and sensor array for detection and analysis of molecular markers in various samples by detecting the change in frequency before and after sample introduction using either a dry condition or a wet condition process.

BACKGROUND OF THE INVENTION Sensor Technology

Sensors are analytical devices composed of a recognition element coupled to a physical transducer measuring mass or optical, electrochemical or thermal properties for qualitative and quantitative detection of analytes.

Sensors have wide applications in: medical, food, industrial, environmental and biodefense areas.

The increasing trend in healthcare strives for early detection for disease screening and prevention. The increasing rate of obesity and the alarming rise in the rate of diabetes in the industrialized world is driving a need for sensors to monitor diabetic patients' glucose levels, increasing pollution, and food safety. This drives the development of sensors for food and environmental applications. The pharmaceutical industry is driving the need for new and rapid sensors to speed up drug discovery rates. The war-on-terrorism is driving the need for new and rapid detection sensors to sense bio-warfare agents for military and civil defence applications. While the demand for sensors in each of the above five areas increases at a high annual rate, the applications in the medical area overshadows the other seemingly important application areas.

Piezoelectric Quartz Crystals

Piezoelectric quartz crystals have been developed as biosensors for the detection of DNA, protein, virus, bacteria, living cells toxin as described in an article by Marx, K. A. Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface, Biomacromolecules, Vol 4(5), 1099-1120, 2003. This technology is applied in biochemical, environmental, food and clinical analyses for various hydrocarbons, gas-phase analytes and pollutants. Numerous combinations of piezoelectric devices with immunological elements have been reported in Yuan, J.-B., Tan, Y.-G., Nie, L.-H., Yao, S.-Z., 2002. Piezoelectric quartz crystal sensors based on ion-pair complexes for the determination of cinchonine in human serum and urine. Anal. Chim. Acta 454, 65-74; and Su, X.-D., Chew, F.-T., Li, S. F. Y., 2000. Piezoelectric quartz crystal based label-free analysis for allergy disease. Biosensor. Bioelectric. 15, 629-639. Nevertheless, as the sensitivity of piezoelectric sensors depends on the oscillating frequency and the area and thickness of the material, there are technical difficulties in producing thinner, smaller quartz devices with higher frequency. These physical barriers limit the application of quartz crystal sensors due to their low sensitivity and high cost.

Moreover, it is noted that in an article entitled “Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface,” Marx, K. A., Biomacromolecules, Vol 4(5), 1099-1120, 2003, an electromechanical quartz crystal microbalance QCM is described. However, quartz microbalances have the sensitivity problems noted above, and as compared with the subject ceramic-based product, the differences are as follows:

Parameters Ceramic Quartz Sensing material Ceramic resonator Quartz crystal Frequency 500 kHz-1 GHz 10 MHz Sample for detection Protein/DNA/carbohydrate/ Not specified different biological markers etc. Markers to be screened Multiple Multiple for certain model no. Max number of markers to 16 4 to 8 be detected simultaneously Measuring condition Protein-protein interaction/DNA Electrochemical reaction hybridization/ligand receptor etc. Dry and wet Liquid Single measurement Not specified Measurement Resonance frequency/impedance/ Potentiostats/Galvanostats resistance/capacitance change Sample volume required Little (few μl) Large (several ml) Sample detection range ng-μg ng-mg Detector size Small Large Sensor Disposable Reuse

Note that the quartz device measures electrochemical change of the sample. In this system, the quartz crystal is sealed to the side opening in a measuring cell wall. Cell volumes of 3.5, 5, 10, 25, 50, and 100 mL are available. In one embodiment of the quartz device, the measurement is under liquid condition that depends on an acoustic wave measurement rather than a resonance frequency change measurement as will be described for the subject system.

As described in WO 91/05261 entitled “Assay method for biological target complexes on the surface of a biosensor”, this is a reference which describes the use of A/F cut thickness shear mode quartz crystal as the sensing material for sample detection. The detection depends on acoustic wave propagation. However, due to the sensitivity problems noted above, it uses complicated steps for analyzing the presence of a target in the sample through indirect measurement of mass changes. The capturing molecule is immobilized on the sensor surface which is a general capturing molecule e.g. avidin for capturing different molecules containing biotin. The biotin containing molecule (the bifunctional conjugate) could be specific antibodies to different analytes. The bifunctional conjugate would bind to the analyte and be captured by the capturing molecule on the sensor surface. To detect a signal, a substrate that would lead to color change due to the interaction between the capturing molecule and the bifunctional conjugate would be added that would be deposited on the sensor surface which could lead to a frequency change. This setup is used due to the low sensitivity of the setup for direct mass detection that would require amplification using a signal generating product deposit on the sensor surface. Besides, the complicated procedures described by WO 91/05261 may not be directly applicable in clinical diagnostics, as simplicity, speed, high through put and low cost are desirable.

On the other hand, piezoelectric ceramics offer the advantage of high ruggedness and ease of fabrication in complex shapes, as well as flexibility due to the higher frequencies at which they can operate and due to their reduced cost. However, the application of ceramics to biological sample detection is limited.

Piezoelectric Ceramic-Based Biosensors

Note that piezoelectricity is a phenomenon exhibited by noncentrosymmetric crystals whereby an electric polarization or charge is induced in the material upon the application of a stress. Piezoelectricity exists in some naturally occurring crystals such as quartz, but the bulk of the piezoelectric materials come from synthetic polycrystalline ferroelectric ceramics, such as lead titanate zirconate (PZT). The coupling of electrical and mechanical energy makes piezoelectric materials useful in a wide range of applications including sensors and actuators, telecommunication, and energy conversion such as described in Setter N ed., “Piezoelectric materials in devices”, Ceramics Laboratory, EPFL, Switzerland, 2002.

For the sensors using ceramic materials, most of the applications describe systems that utilize the ceramic material as an underlying material for coating another sensing material. Moreover, the measurement depends on electrochemical changes rather than utilizing the ceramic material's piezoelectric property that enables direct frequency measurement.

For ceramic biosensor applications, the cantilever or the acoustic property of the material has been utilized, rather than the direct measurement of resonance frequency change for sample measurement. See Atonomics APS, Microsensors and method for detecting targeting analytes, WO 2002/020832, 14.03.2002; Drexel University, Piezoelectric cantilever sensor, WO 2005/043126, 12.05.2005; and Valtion Teknillinen Tutkimuskeskus, Micromechanical Sensor, Sensor Array and Method, WO 2007/006843, 18.01.2007 and Atonomics APS, Surface Acoustic Wave Sensor Comprising a Hydrogel, EP 1804059, 04.07.2007.

For the sensors using ceramic materials, most of the above publications describe systems that utilize the ceramic material as an underlying material for coating another sensing material. Moreover, the measurement depends on electrochemical changes rather than utilizing the ceramic material's piezoelectric property that enables direct frequency measurement.

More than 150 publications related to piezoelectric biosensors have been published, while only a few publications are related to the utilization of the ceramic piezoelectric property in biosensor applications. These are articles by Lee J H, Hwang K S, Park J, Yoon K H, Yoon D S, Kim T S. Immunoassay of prostate-specific antigen (PSA) using resonant frequency shift of piezoelectric nanomechanical microcantilever. Biosens Bioelectron. 2005 Apr. 15; 20(10):2157-62; Verissimo M I S, Mantas P Q, Senos A M R, Oliveir J A B P, Gomes M T S R. Suitability of PZT ceramics for mass sensors versus widespread use of quartz crystals. Sensors and Actuators B 2003; 95:25-31; and Eklund A, Backlund T, Lindahl O A. A resonator sensor for measurement of intraocular pressure--evaluation in an in vitro pig-eye model. Physiol Meas. 2000 August; 21(3):355-67.

For instance, with respect to EP 1 804 059, what is described are acoustic wave sensors that detect materials by generating and observing an acoustic wave. The detection is based on the acoustic wave that propagates through or on the surface of the material (in this case by surface acoustic wave) that causes changes along the propagation path to velocity and/or amplitude of the wave. The SAW sensor design has interdigitized gold electrodes constructed on a piezoelectric support comprising lithium tantalite, with each sensor being coated with SiO₂ for electric insulation and chemical attachment. The recognizing element is embedded in a hydrogel and is attached to the sensor surface after a polymerization process. The amplitude, frequency, and/or phase characteristics of the sensor are measured and correlated to a corresponding physical quantity that is a result of the application of an analyte. The application of the sample containing analyte leads to a change of both mass and a property of hydrogel that causes the frequency change.

As to WO 02/20832, this reference describes the use of microscopic flexible mechanical structures such as micro-cantilevers or micromembranes to provide a non-fluorescent sensor. Binding of the analyte induces stress in the microsensor which results in deflection of the microsensor. T he deflection is detected by a change in an electrical parameter of piezoelectric element e.g. voltage, resistance and current. The detection is based on the following principle: as the cantilever scans across the indentation of a polymer film, the cantilever deflects along the indentation of the film, causing stress on the PZT film. The variation of stress in the PZT film produces self-generated charges on the surface of the PZT capacitor without applying a voltage. These charges are not generated by the absolute stress, but by the variation in the stress in which the variation of the stress leads to frequency change. Variations in stress measurements are however different as a sensing mechanism from direct absolute mass resonance sensing that is the phenomenon used in the subject sensor.

With respect to the Verissimo reference, according to the information in this article, the preparation of PZT used is Nb—Mn-PZT. The ceramic disc has a relatively low frequency range of 100-200 kHz, and it has been found that the viability of such a low-frequency system for use as a biosensor is questionable.

As to the Eklund et al. system, a PZT rod having dimensions of 25×5×1 mm is used as the sensor element for measuring pressure change through the frequency shift between unloaded and loaded conditions. The unloaded resonance frequency is approximately 82.9 kHz and the measuring range is 200-700 kHz. The PZT rod used is much larger in size than the subject sensor and detection is in the low frequency range, not comparable to the ceramic resonators described herein and not suitable for development into an array of sensors for multiple sample measurement.

Finally, with respect to the utilization of PZT, Jong Hun Lee et al., in an article entitled “Immunoassay of prostate-specific antigen (PSA) using resonant frequency shift of piezoelectric nanomechanical microcantilever” Biosens Bioelectron, 2005 Apr. 15; 20(10:2157-62, describes a PCT cantilever device that measures resonant frequency change. In a piezoelectric cantilever system such as the PZT cantilever device of Jong Hung Lee et al., the sensing mechanism involves stress measurement in terms of frequency change. The nanomechanical PZT cantilevers are homemade with a resonance frequency of 16 kHz or 61 kHz depending on the size of the PZT used in the experiment and the measurement range is from 100-400 Hz. However the relatively low frequencies used present problems.

If a biosensor platform could be developed using a piezoelectric resonator that operates at high frequency and uses resonant frequency change as the core component of detection, the platform could be applied to detection of various biomarkers in different fields of application. As clinical applications have the greatest share in the biosensor market and because cancer is the leading cause of death worldwide, biosensors for detection of multiple cancer markers address many market needs.

Cancer is a Global Healthcare Problem

Cancer is the uncontrolled growth and spread of cells that may affect almost any tissue of the body. More than 11 million people are diagnosed with cancer every year. It is estimated that there will be 16 million new cases every year by 2020. Cancer causes 7.6 million deaths every year, or 13% of deaths worldwide. Cancer is now the leading cause of death in China and Hong Kong whereas malignant neoplasms lead to a death rate of 23.92% in China in 2004 and 31.8% in HK in 2005 respectively.

Lung, colorectal and stomach cancer are among the five most common cancers in the world for both men and women. Among men, lung and stomach cancer are the most common cancers worldwide. For women, the most common cancers are breast and cervical cancer. In China, approximately 1.3 million people die of cancer each year. The main cancer sites are stomach, liver, lung, and oesophagus. These four cancers account for 74% of all cancer deaths in the country. Among all deaths from cancer, lung cancer is the most common, accounting for 22.0% of all cancer deaths for males, followed by stomach cancer with 17.8% and hepatic cancer with 13.0%. Stomach cancer is the most common for females with 14.8%, followed by lung cancer with 12.7% and colon cancer with 10.1%. Stomach cancer and cervix/corpus uteri cancer which used to be major causes of death among the Japanese now show decreasing trends, indicating the advance of medical technologies such as early detection and early treatment is also a relevant factor.

Cancer is largely preventable. Around one-third of all cancers are preventable by stopping smoking, providing healthy food and avoiding the exposure to carcinogens. Some of the most frequent cancer types are curable by surgery, chemotherapy or radiotherapy if early detection is available.

Cancer Diagnostic Methods

Throughout the years, researchers have been working hard in developing various methods for diagnosing cancer. Most of the cancer diagnostic assays depend on biomarker detection for diagnosis, staging, prognosis and management of patients with cancer. Biomarkers are substances that can often be detected in higher-than-normal amounts in the blood, urine, or body tissues of some patients with certain types of cancer. Cancer markers are produced either by the tumour itself or by the body in response to the presence of cancer or certain benign (noncancerous) conditions.

Note that most of the immunoassays only target a single cancer marker.

Need for Development of New Cancer Diagnostic Technology

Cancer diagnostics are a naturally growing market, owing to the increased incidence of cancer and an aging population. An aging population will lead to increased number of cancer patients. Unhealthy lifestyles are another cause for increased cancer incidence, such as smoking, poor or imbalanced diet, inactive lifestyles, and increased exposure to ultraviolet light and polluted environments.

Most diagnostic assays are based on immunoassays (such as ELISA) which are widely available and are routinely employed in clinical laboratories. Normally a single marker is detected by each ELISA kit. For multiple antigen detection, several ELISA kits have to be used. In addition, these methods usually require a radioisotope, an enzyme, fluorescence or a colloidal gold-labeled antibody or antigen, and may suffer from drawbacks of requiring skilled personnel, time-consuming procedures and expensive chemicals.

Many healthcare systems in developed countries have strategies to improve cancer care and survival rates through improvements in cancer screening, with the emphasis on early detection. Recent advances in medical research have revealed that cancers are caused by many factors and each type of cancer can be represented by multiple cancer markers. Existing methods for cancer marker detection are based on immunoassays, which detect one protein marker at a time and are unsuitable for clinical applications that require the ability to determine multiple markers in a timely and cost-effective manner. There is a significant and pressing need for new cancer diagnostic technologies for parallel detection of multiple cancer markers and especially a piezoelectric ceramic cancer diagnostic biosensor for simultaneous detection of multiple cancer markers.

The cancer diagnostic market lies in two areas. The first lies in disease management and monitoring to observe therapeutic response and signs of recurrence. The second is screening that targets people with increased risk for cancer because of family history, occupation, or age. A biosensor array can be used in the targeting cancer screening area for fast screening of high risk groups. It can also be used to monitor the effectiveness of treatment or recurrence of cancer after therapy.

Thus, there is a need for a sensor platform for the simultaneous detection of multiple markers for, for instance, not only cancer detection but also heart disease detection and prognosis, food safety evaluation, environmental toxin monitoring and other applications.

SUMMARY OF THE INVENTION

The present invention relates to a sensor for detection of a marker in a sample that utilizes a high-frequency 500 kHz-1 GHz piezoelectric ceramic resonator sensor and measurement of resonant frequency change.

In one embodiment, the system described herein uses a thickness extensional (TE) mode piezoelectric ceramic resonator as a sensing material. The capturing molecule which is specific for the analytes is immobilized on the sensor surface for capturing the analyte and detection of mass change is done directly through resonance frequency change. For high sensitivity sample detection, a high frequency probing of the piezoelectric material is required. This is possible as high frequency piezoelectric ceramic resonators can be easily manufactured for sample detection, whereas quartz crystals cannot. Thus, the high frequency of the subject provides a sensor that system is sensitive enough to detect a low analyte level that provides valuable information for clinical applications. Besides, TE mode ceramic resonators (poled in the thickness direction) are more effective for very flexible structures when compared to thickness-shear mode ceramic resonators (poled in the longitudinal direction) that are more effective for stiffer structures. IE mode ceramic resonators are also uniquely suitable for array type sensor development due to their compact size and low cost. In addition, the TE mode provides uniform displacement along the thickness direction thus providing uniform measurements. As a result, the subject sensing material or sensing mechanism is different from that described in other publications in which quartz, cantilever or acoustic wave detection mechanisms are described.

The system uses neither a surface acoustic wave sensor nor a hydrogel located on the surface of the sensor in which a molecular recognition component is immobilized as disclosed in EP 1 804 059 A2. The detection of the sample according to the subject invention is based on the direct resonance frequency change of the piezoelectric ceramic resonator sensor due to sample application. The detection of the analyte is directly due to the interaction of the capturing molecule on the surface of sensor and the analyte present in the sample that leads to mass change which is reflected by the resonance frequency change. This is not due to the interaction that affects the propagation of wave through a hydrogel as described in EP 1 804 059.

The sample detection sensor units are arranged in a single or an array format. In one embodiment, multiple molecular markers are simultaneously monitored by driving different resonators with different frequencies. Typically the sensor is mounted on a printed circuit board through a standard soldering technique and is connected to an oscillator. In one embodiment, the sensor includes two piezoelectric resonators aligned in a series and separated by a gap, with one of the resonators being the reference resonator and the other resonator having a coating layer on the electrode surface with a capture molecule for the marker being immobilized thereupon.

The ceramic material of the resonator is preferably a synthetic polycrystalline ferroelectric ceramic, preferably lead titanate zirconate (PZT). Gold electrodes are constructed on the ceramic material through direct sputtering, and there is no need for an SiO₂ deposition (as described in EP 1 804 059) on top of the gold electrode before capturing molecule attachments for sample analysis. The capturing molecule is immobilized directly on the gold electrode surface of the sensor through a linker group which is used for sample detection to measure the mass change directly.

In one embodiment, the sensor includes a piezoelectric ceramic sensor with capture molecules for markers and a sample loading cartridge. In a further embodiment, the sensor additionally includes means for recording the binding interaction between the capture molecule and the marker; and means for data analysis and correlation of the measured marker frequency differential to the normal frequency shift in the sample. The analyzed samples include for instance blood, serum, plasma, urine or any non-organic solution depending on the markers to be detected. The samples are used for disease diagnosis, or other applications such as food analysis, environmental pollution analysis and biodefense applications. The present invention addresses several important technical and practical issues, namely the technical feasibility of using piezoelectric ceramic resonators as a central component of sensor devices, and the technical feasibility of applying ceramic-based sensor devices in molecular diagnostics.

The present invention has several important applications including disease diagnosis and in particular, cancer detection in humans. In one embodiment, the subject technique offers simultaneous detection of multiple cancer markers with simple, fast and high throughput operation, and at a reduced cost. The biosensor platform established is used for development of a series of products for other applications including, but not limited to cardiovascular disease diagnostics, autoimmune disease diagnostics, coagulation disorder diagnostics, and food analysis.

More particularly, tumor markers are used to detect and analyze the tendency of cancer development using a sensor that includes a high frequency piezoelectric ceramic resonator. Sometimes the tumor marker molecules will be referred to herein as a tumor “biomarker” or a similar term to denote a physiological origin for these molecules. Preferred use of the invention generally involves detection and quantitation of markers that have been found to be indicative of the presence or tendency of disease development.

For example, the presence or the tendency of cancer development is determined with high sensitivity and selectivity by detecting and quantifying the tumor markers. The preferred use of the subject invention is via a bench top sensor that is connected to a computation system, thereby facilitating a simple and convenient test of samples by technical staff in a hospital or by doctors at clinics. The sensor and sensor array described herein is well suited to the simultaneous detection of multiple cancer markers and development of sensors for other disease diagnostics such as cardiovascular diseases, autoimmune diseases, coagulation disorder, food safety analysis. Thus, with the use of the subject system, the opportunities for early medical intervention are increased.

In particular, the technology platform consists of a piezoelectric ceramic sensor with capture molecules for markers, a sample loading cartridge, a computation system for signal output and analysis for monitoring marker levels in samples such as blood, serum, plasma, and urine. The sensor array is used for simultaneous detection of multiple markers including but not limited to detection of multiple cancer markers for screening and early diagnosis of the most common types of cancers; detection of multiple heart disease markers for early diagnosis and prognosis of heart disease; detection of multiple food contaminants for improvement of food safety; detection of multiple environmental toxins for monitoring environmental pollution and detection of multiple bio-warfare agents for military and civil defence applications.

In a further embodiment, the invention provides a technology platform with a sensor including a surface substrate for immobilization of a first molecule that specifically binds a second molecule; a sample application cartridge containing a sample pad or microfluidic channels for sample loading; a means of detecting the binding interaction between the capturing molecule and the marker in the sample in terms of a frequency shift, and means for evaluating and correlating the binding of the molecules to the tendency of disease development.

More particularly, the present invention also relates to a method for detection and evaluation of the amount of one or more markers in a sample using the subject sensor or the sensor array, wherein the resonance frequency change before and after a sample application is determined and correlated to the mass of the marker being captured by the capture molecule on the ceramic resonator surface. Specifically, the subject method includes applying an oscillating electric field across the sensor, measuring at least one resonance frequency of the sensor as the pre-sample application resonance frequency, providing the sample through a sample loading cartridge and incubation or produce an interaction of the capture molecule and the marker, applying an oscillating electric field across the sensor, measuring at least one resonance frequency of the sensor as the post-sample application resonance frequency, and correlating the frequency change before and after sample application to the surface mass and/or surface density according to a default standard range, thereby detecting and analyzing the output of one or more biomarkers. In a preferred embodiment, a computational system correlates surface mass and/or surface density with the amount of marker bound by the capture molecule.

In another preferred embodiment, the sensor is sensitive to minute differences in measurement of mass. Preferably, the sensor comprises a piezoelectric ceramic resonator as a surface substrate for immobilizing a first molecule. The principle components of the sensor system include a piezoelectric ceramic resonator sensor, an oscillator and a control circuit. In the piezoelectric ceramic resonator sensor there are typically two carefully matched ceramic resonators coupled in series and separated by a small gap. Carefully matched ceramic resonators are also aligned in series and are separated by a small gap. These resonators are arrayed in a square format for multiple biomarker detection. Both ceramic resonators in the case of single marker detection and all the ceramic resonators in the case of multiple marker detection are exposed to the sample, whereby one of the ceramic resonators is used as a reference resonator for environmental control. The difference in frequency of pre and post sample application using a reference resonator frequency is the frequency change due to the marker interaction. This is a very sensitive indication of the mass being deposited on the ceramic resonator surface. The frequency change is proportional to the mass of the molecule being captured by the capture molecule on the sensing area. This frequency change is electronically recorded and transferred to a computer for data analysis.

In another embodiment, the sensor platform includes an integrated circuit for sensor control, signal recording and conditioning, temperature monitoring and a microcontroller for data acquisition and data formatting.

In another embodiment, the ceramic resonator biosensor includes an integrated circuit for measurement of sensor's resonant frequency over a narrow range of impedance.

Other examples of ceramic resonator sensors include, but are not limited to ceramic resonator biosensors that are used to measure the mass of a substantial drop of the target molecule contained in a sample solution, with the measurement being carried out in the gas phase. Thus, there is no need for the correction of viscous damping losses.

In another preferred embodiment, ceramic resonator sensors include an integrated circuit to measure, in addition to determining mass, the molecular species of the material deposited on the ceramic resonator sensor's reaction center.

In one aspect, the amount of each marker is measured in the sample as the resonant frequency change before and after sample loading. The mass change represented by the resonant frequency change is correlated to a default standard mass range. An increase in the frequency change indicates the amount of marker presented in the sample and gives an indication relative to the development or tendency of development of cancer or other types of disease as compared to clinically relevant data. Monitoring which markers are detected and at what levels provide specific information on disease type and the tendency for the development of a particular disease that could provide a chance for early treatment.

In another aspect, a single marker is used in combination with one non-specific marker to the target molecule for monitoring the development or tendency of development of a particular cancer or other diseases. Preferably, multiple markers are used in combination with one non-specific marker to the target molecules in the sample for simultaneously monitoring the development or tendency of development of cancers or other diseases.

In another embodiment, a stable sensor-oscillating system is included for sensitive detection of molecule interaction on the surface of the ceramic resonator.

In another embodiment, data is generated on immobilized samples on a ceramic resonator surface, after which changes in mass are detected. Preferably, the ceramic resonator sensor includes an integrated circuit to measure, in addition to determining mass, the molecular species of the material deposited on the ceramic resonator biosensor's reaction center. The data is transformed into computer readable form; and an algorithm is executed that classifies the data according to user input parameters for detecting signals representing the marker level contained in a sample indicating the development or the tendency of development of a particular disease.

In another embodiment, the presence of certain biomarkers is indicative of the development or tendency of development of different cancer types. For example, detection of one or more tumor markers at a certain level is indicative of the development or tendency of the development of cancer.

Preferred methods for detection and diagnosis of cancer include detecting at least one or more markers in a sample, and correlating the detection of one or more markers with the development or tendency of development of cancer, wherein one or more protein markers are selected from tumor markers such as Alpha-Fetoprotein (AFP); Cancer Antigen (CA): CA 19-9; CA 27.29; CA 125; Carcinoembryonic Antigen (CEA); beta subunit of human chronic gonadotropin (β-hCG); and Prostate-Specific Antigen (PSA).

According to the subject invention, the sample is preferably a gas or a fluid, for example, blood, serum, plasma, urine. The immobilized capture molecule is a DNA probe, an RNA probe, a carbohydrate, a protein, an antibody, a fragment, a variant or derivative thereof or any other molecule that specifically binds at least one or more of the markers, preferably, but not limited, tumor markers.

In another embodiment, the sample is any non-organic solution, a gas containing the target molecule, for example, a gas or a solution containing food components, a polluted water sample in which the agent is an antibody, DNA, or another molecule that specifically binds at least one or more of the markers.

In another embodiment, the sample is loaded through a sample loading cartridge, wherein a sample loading pad or microfluidic channel is embedded.

When multiple sensors are used in a microsensor array, each resonator is driven with a slightly different frequency to permit differentiation.

The frequency range of the ceramic resonators is the same. However, to prevent interference between the resonators when they are arranged in an array format, the frequency for the adjacent resonators have a frequency difference of 1 MHz (e.g. adjacent resonators would have frequencies: Resonator 1 with frequency of 40 MHz and Resonator 2 with frequency of 41 MHz or 39 MHz respectively).

When measuring the interaction of different antigen-antibody reactions, it is the frequency difference pre- and post-sample addition that matters, not the absolute frequency recorded. The frequency difference for different markers could be the same or different which depends on the presence and amount of the biomarker in the sample solution. The specificity of the detection depends on the specific antibody immobilized on different ceramic resonators in the array format, not on the frequency difference. In one embodiment, the frequency difference for pre- and post-sample addition of each marker is compared to a standard in which the frequency change was predetermined by using standard samples.

If one is using microfluidics, or using microfluidic flow for sample application, the measurement can be accomplished in the following mode:

-   -   Dry condition         -   Using microfluidic flow for sample addition, under the dry             condition, the frequency is not measured continuously,             rather two spot frequencies are recorded, one before the             sample addition and the other after the sample addition.             Note the pre-sample addition frequency is measured and             recorded. Then the sample is added using a microfluidic             setup and is washed and dried before measuring the frequency             again. The frequency difference of the two frequencies             recorded is compared to a standard frequency for each of the             markers. A spigot is introduced to control the flow. But if             there is no spigot, the capillary action is good enough to             drive the flow.     -   Wet condition         -   Using microfluidic flow for sample addition, under the wet             condition, the frequency is measured continuously or two             spot frequencies are recorded.         -   Continuous sample addition is achieved through a             microfluidic setup, whereby real time frequency change is             recorded. The flow rate and volume is recorded and the             frequency change is correlated to the predetermined             frequency change of the standard of each marker measured             under the same condition. By using such a set up, the             binding kinetic of the biomarkers is evaluated.         -   Two spot frequencies measurement is determined by sample             application using a microfluidic setup. The frequency before             sample application is recorded. A spigot is used to stop the             flow after a fixed volume sample introduction. Then the             frequency after sample addition is recorded when             equilibration has been reached. The frequency change is             correlated to the predetermined frequency change of the             standard of each marker measured under the same condition.

Note that the piezoelectric ceramic uses PZT. The PZT has a formula of Pbα-aMea[(MII1/3MV(2+b)/3)zTixZr1−x−z]O3, wherein Me represents a metal element; MII represents an acceptor element of a divalent metal element; MV represents a donor element of a pentavalent metal element; and z, b, x, α and a satisfy 0.05≦z≦0.40, 0<bz/3≦0.035, 0.345≦x≦0.480, 0.965≦α≦1.020 and 0≦a≦0.05. Note the ceramic resonator is probed in a high frequency range, ie 50 kHz-1 GHz-100 MHz. The properties of the high frequency resonator used is very different from the low-frequency devices mentioned above.

Ceramics are available at lower cost than quartz, which is a very important consideration for medical diagnostic applications. Versatility is another key feature of the materials, as compositions are selected and modified to achieve a desirable combination of properties. Besides, miniaturization of the ceramic material is possible, which makes it suitable to manufacture protein microarrays. Thus, piezoelectric ceramic resonators are used for sensor platform development in the different applications indicated above.

In summary, a sensor is provided for the detection of a marker in a sample in which the sensor includes a high frequency 500 kHz-1 GHz piezoelectric ceramic resonator, with the system measuring resonant frequency change. In one embodiment, the piezoelectric sensor operates in the thickness extensional (TE) mode, with the high frequency and TE mode permitting fabrication of an exceptionally small size sensor capable of being arrayed in a handheld unit.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the subject invention will be better understood in connection with the Detailed Description, in conjunction with the Drawings, of which:

FIG. 1 is a schematic representation of the sensor concept for detection of molecular markers;

FIG. 2 is a schematic representation of the sensor concept of an assembled sensor array;

FIG. 3 is a schematic diagram of the piezoelectric ceramic biosensor platform;

FIG. 4 is a schematic representation of immobilization for capturing molecule and target molecule detection;

FIG. 5 is a representation of an individual cancer marker sensor device;

FIG. 6 is a representation of a microarray detection unit for simultaneous detection of 16 markers, with each ceramic resonator chip on the PCB board representing an individual sensor unit as shown in FIG. 5, and with the ceramic resonator being connected to the oscillating circuit underneath the ceramic resonator through a slot;

FIG. 7 is a schematic representation of the sample application system using a sample pad for marker detection;

FIG. 8 is a schematic representation of the cartridge for sample introduction using lateral flow;

FIG. 9 is a schematic representation of the sample application system for microarray biosensor detection units using a sample pad for marker detection, with FIG. 9A showing the design and set up of the sample application system; and with FIG. 9B indicating flow direction after sample loading;

FIG. 10 is a schematic representation of the direct sample application system;

FIG. 11 is a schematic representation of the sample application system for microarray sensor detection units for the direct sample application;

FIG. 12 is a diagrammatic representation of a sensing interface that converts the antibody-antigen binding event into an electrical signal; and,

FIG. 13 is a schematic representation of the sample application system using microfluidic channels for markers detection.

DETAILED DESCRIPTION

A novel high frequency sensor is developed using piezoelectric resonators as the core component of the sensor, and a sensor array is developed for simultaneous detection of multiple markers e.g. simultaneous detection of cancer markers for screening and early diagnosis of the most common types of cancers. The sensor technology platform can be used for developing sensors for various applications in different areas including but not limited to the diagnostic field.

The subject technology platform has a wide application for development of different types of sensors. T he sensor thus developed has competitive advantages in that it provides specific and sensitive detection of target molecules with a simple procedure at high throughput and reduced cost.

Prior to setting forth the invention, the following definitions are provided. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

“Biomarker”, “marker” or “target molecule' are used interchangeably herein, and in the context of the present invention refers to a protein or other molecules which is or are differentially present in a sample taken from patients having or having the tendency of developing cancer or other kind or diseases or are differentially present in a food sample containing pathogens, or in a water sample containing pollutants or in a gas sample containing different gases as compared to a sample taken from control subjects (e.g. healthy subject, clean food, unpolluted water solution or control gases)

“Multiple” refers preferably to a group of at least two, preferably, at least 16 members. The maximum number of members is unlimited, but at least 100 members are within the scope of the subjection invention.

“Antibody” or “capturing molecule” are used interchangeably herein and refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes includes the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes. Antibodies exist, e.g. as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g. Fab′ and F(ab)′₂ fragments. The term “antibody”, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies.

“Diagnosis” refers to the identification of the presence or nature of a pathological condition.

“Immunoassay” is an assay that uses an antibody to specifically bind an antigen (e.g., a maker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

“Immobilization” refers to the fixation of capturing molecules on the surface of a substrate using different chemical or biological methods.

The phrase “specifically binds” refers to a binding reaction that is determinative of the presence of a target molecule in a heterogeneous population of proteins and other components in the sample. Thus, under designated sensor measurement condition, the specific antibodies bind to a particular target molecule at least two times the background and do not substantially bind in a significant amount to other components present in the sample.

“Detect” refers to the identification of the presence, absence or the amount of the target molecule in the sample.

“Sample” refers to any non-organic solution containing a target molecule. A sample may be blood, plasma, serum, urine, food containing solution, water containing pollutants, and gases amongst others. The target molecule contained in the sample may be polypeptides, peptides, proteins, antibodies, cells, chemicals, and gases amongst others.

“Default standard” refers to the standard curve representing the frequency change at each concentration of the antigen using standard antigens for testing.

“Sensor” refers to a small, portable, analytical device based on the combination of capturing molecules with an appropriate transducer and which detects chemical or biological materials selectively and with high sensitivity.

The phrase “single unit” refers to a biosensor setup that is used for the detection and analysis of a single marker.

“Microarray” refers to a sensor setup that incorporates several single unit ceramic resonators together for the simultaneous detection of multiple markers.

The phrase “Lateral flow” refers to the sample application technique utilizing a sample pad, whereby the sample containing the target molecule is loaded onto the sample pad and capillary action drives the solution flow from the sample pad to the direction of the absorption pad.

“Microfluidic” refers to a sample loading technique utilizing small channels fabricated on a minute area using photoresist technology.

The phrase “development or tendency of development” refers to the status of the patient for the likelihood of developing a certain disease based on the result obtained from the subject sensor.

The phrase “high throughput” refers to a large number of samples that can be processed in a given unit of time.

Sensor Concepts for Marker Detection

The subject sensor technology provides a unique opportunity to develop diagnostic products for detection of various biomarkers. The subject sensor concept includes a sensing interface comprising the specific antibody against a selected marker e.g. cancer markers (the antigen), and a transducer (such as ceramic resonator) that can convert the antibody-antigen binding event into an electrical signal.

As illustrated in FIG. 1, what is shown is a biosensor 10, a sensing interface 12, biomarkers 14, other reactants in a sample 15, specific antibodies 16, a bio/chemical interface 18, a piezoelectric ceramic resonator 20, frequency measurement 22, signal conversion 24 and an output to a computation system 26.

Referring now to FIG. 1, a biosensor 10 includes a sensing interface 12 in which a specific antibody is immobilized to the piezoelectric ceramic resonator 20 through bio/chemical interface 18 that reacted with a selected biomarker. In this case biomarkers 14 reacted with specific antibodies 16, whereas other reactants 15 in the sample could not. Piezoelectric ceramic resonator 20 that is connected to an oscillator acts as a signal transducer for frequency measurement 22, and the resultant resonance frequency change is outputted to computation system 26 after signal conversion 24.

An individual sensor unit for detecting a single marker is developed first and is assembled together in an array-format to produce a sensor array that is capable of simultaneously detecting a panel of markers as illustrated in FIG. 2. In FIG. 2 what is shown is an individual biosensor unit 30, individual biosensor units in an array format 32, a biosensor array assembled on a data acquisition card 34 and a data acquisition card inserted in a portable device 36 e.g. a mobile phone for simultaneous detection and analysis of a panel of markers.

Array-based immunoassays involving effective screening of antigen-antibody interactions using smaller amounts of samples can play an important role in screening markers with reduced cost. Results to date show that the microarray test format provides an equivalent performance to ELISA, indicating that multi-parameter tests can be incorporated for routine use in clinical laboratories. It also offers a significant advantage in convenience, throughput and cost when compared to traditional test formats.

By using the subject sensor array as the diagnostic tool, multiple markers can be detected at the same time. Miniaturization also enables the integration of multiple arrays for screening multiple patient samples. The subject biosensor array has the advantages of low sample consumption with improved quantitative accuracy, sensitivity and speed.

Piezoelectric Ceramic Sensor: Technical Considerations

Technological change in the sensor area has been driven in part by the successful development of home-use glucose sensors. For the piezoelectric ceramic sensor products, the performance characteristics and design requirements related to the use of sensors in diagnostic test systems, and the functional requirements and processing technologies needed to bring this new technology into the diagnostic marketplace are outlined below.

(1) Inherent Performance Characteristics

The subject sensors offer performance characteristics that would make them well-suited for applications in critical care, point-of-care, bedside, field-use and home-use test systems. The ceramic resonators are inherently smaller, more responsive, and more versatile than quartz crystals. When coupled with molecules, they can be formed into sensors with high sensitivity and specificity; and by adapting manufacturing processes from the semiconductor industry, they can be produced at unit costs low enough to make their use cost-effective. The key characteristics that make the sensors attractive are the following:

Small Size. To achieve the portability necessary for developing true handheld, bedside, field and home-use testing systems, miniaturization is a must. In some cases, personal convenience calls for a handheld, battery-powered unit. In others_(;) the fact that handheld units can easily be dropped or misplaced is considered a disadvantage, making a bench top unit more desirable. The small size of ceramic sensors is an advantage in many such applications, offering a platform that can be readily adapted to a variety of formats.

Time-to-Result. Reductions in the size of test instruments may increase the number of settings where they can be used, but time-to-result is also a key criterion for measuring the acceptability of any such test system. Since the sensors require smaller sample volumes than traditional analyzers, they can potentially have a faster reaction time. How ever, the reaction time of a sensor is generally determined by the speed at which the sample diffuses, and the speed of the molecular interactions between the analyte and the sensor's recognition element.

Versatility. The subject sensor technology offers a universal platform for developing other diagnostic systems for many analytes e.g. clinical, food, environmental etc. Like their larger clinical laboratory counterparts, these sensors will inevitably compete to some degree on the basis of their menu of available tests. Using these technologies could enable manufacturers to expand their test menus. The subject sensor can be applied for a wide variety of analytes, including chemistries, immunoassays, and nucleic acid assays.

Sensitivity and Specificity. The subject sensors using high-sensitivity high-frequency piezoelectric transducers and high-specificity immunoassays to achieve the sensitivity and specificity are required in diagnostic test systems. The subject sensor reliably detects a wide range of markers with sensitivity and specificity suitable for clinical diagnostic testing e.g. cancer diagnostic, food contaminant detection, and environmental pollutant detection.

Ease of Manufacturing. The subject sensors enable the production of low-cost sensors in large quantities using the MEMS processing techniques developed by the semiconductor industry for the production of the sensors. One stumbling block may be the incompatibility of such techniques with the processing requirements of unstable biological compounds. However, this difficulty is overcome with recently developed bio-dotting (microarraying) liquid dispensing techniques, such that the promise of low-cost, easily manufactured sensors can be fulfilled.

Cost-Effectiveness. The actual cost of the ceramic sensor components is probably of less importance than the cost of high-quality antibodies. Nevertheless, the well established immunoassay industry should provide great flexibility in sourcing the biological components cost-effectively. In addition, advances in the sensor design and manufacturing should further reduce the costs associated with using them.

(2) System Requirements

Although the sensors may inherently possess a number of characteristics that would make them useful in different applications, especially diagnostic applications, FDA and other regulatory agencies worldwide will have some influence over the eventual shape of any diagnostic test system that incorporates sensors. Users also impose performance requirements for such systems, and these requirements can vary widely according to the type of user, the settings in which the system is designed to be used, and the actual types of tests the system will run. Taken together, these additional system requirements pose a considerable challenge for the sensors. Following are some of the key areas in which regulatory or customer requirements may be important.

Precision and Accuracy. Clinical laboratories are accustomed to the precision and accuracy delivered by today's highly sophisticated laboratory equipment, and they are unlikely to tolerate any reduction of these parameters. The same standards are also being applied to test systems designed for use outside the clinical laboratory, meaning that even units designed for home use will be required to perform as precisely and accurately as much larger systems. This requirement is a challenge to the design of sensor systems that can be operated reliably without the intervention of trained technicians.

Use and Shelf Life. The shelf life of the sensors can be a key factor in achieving efficiencies in manufacturing, distribution, and sales. Shelf life is even more important for sensors designed for the over-the-counter or prescription home-use market. The challenge is to design sensor components, particularly the biological components at the sensor interface, to maintain the integrity of minute amounts of chemical or biological components for several months, preferably at ambient temperatures. The biological interfaces must also remain stable in use.

Ease of Use. The sensors diagnostic systems should be easy to use. The challenge is to reduce the operational complexity of the system, which is mainly determined by the user interface. The microarray format of the subject sensors can help to reduce the complexity of the user interface through automated calibration or quality control using one or some of the arrayed units.

Whole Blood. Use of whole blood directly eliminates the need for extensive sample preparation. For all sensors, achieving this goal may be one of the most challenging tasks of all. This is largely because of the relatively wide spectrum of pH, ionic strength, viscosity, water content, and interfering substances that can be present in a whole blood patient sample. These factors can affect the response of the molecules used in the sensor, thereby reducing the accuracy and performance of the system.

Development of the Subject Piezoelectric Sensor

In a preferred embodiment, the specific capturing molecule e.g. antibodies (the recognition elements) against the selected markers is immobilized on piezoelectric ceramic resonators for detection of markers (antigens or analytes) presented in samples e.g. patient samples, food samples, water samples, and gas samples. Signals generated by the ceramic resonator including the oscillating frequency may be modulated when the resonator-antibody interface undergoes certain changes due to the binding of the antigens to the immobilized antibodies. A sensor unit for detecting a single marker is developed to evaluate the feasibility of the sensor for marker detection. This was followed by the development of an array-format sensor device that is capable of simultaneous detecting a panel of markers.

The development of piezoelectric ceramic biosensor was as follows: The property of the ceramic resonator was considered and the measurement parameters in terms of oscillating frequency, impedance and capacitance was developed. This was followed by selecting the suitable surface chemistry for the selected material e.g. ceramic metal, epoxy, plastic etc. Then the sensing mechanism was validated through mass, density viscoelasticity, dielectric measurement. When these parameters were determined, the sensing interface was constructed through self-assembly, sol-gel or multi-layer technologies which can be used for marker e.g. cancer detection. The suitable antibodies were screened and the binding format and sensitivity was determined. Having developed the individual sensor unit, these units were assembled into an array format and normalization, specificity and clinical validation procedures were performed. After validation, development of the ceramic resonators was complete.

As an illustrative example, not meant to limit or construe the invention in any way, the following is provided. Sensors for the diagnostic of cancer markers are used as an example for the sensor technology platform development. High-throughput sensor arrays are developed for fast patient sample screening based on the platform developed. The platform developed can also be modified to increase the number of cancer markers per array unit to increase the detection spectrum of cancer types. More tumor markers can be included when other organ specific tumor markers are identified. The platform developed can also be used for the development of sensors for markers detection in different applications such as cardiovascular disease, diabetes, food contaminants, environmental pollutants etc.

Sensor Design

The development of the sensor for detection of different types of markers involves the utilization of the piezoelectric property of ceramic material, the specific interaction between antibodies and antigens, and the measurement of the frequency (and/or other parameters) changes due to antibody-antigen interactions.

FIG. 3 shows the overall design of the microarray sensor, namely a ceramic sensor 40, a ceramic resonator 42, a data acquisition card 44, a computer with data analysis software 46, a thermal controller 48, a UPS 50, a single unit sensor 52 and an oscillator 54.

Referring to FIG. 3, in one embodiment of the subject system, ceramic sensor array 40 involves the use of ceramic resonator 42. The output of the resonator is coupled to data acquisition card 44 which is in turn coupled to computer 46 that is programmed with analysis software. Not e that thermal controller 48 is used in the system for temperature control and all the modules are powered from UPS 50. Note also that a single unit 52 of ceramic biosensor array 40 shows the resonator configuration of the subject invention that is connected to oscillator 54.

The system is composed of multiple subunits in an array format, each subunit containing a different marker detecting component that can be addressed individually, either in parallel or in series. A thermal controller may be included to control reaction temperature. The recorded frequency change during marker detection is sent to a data acquisition card and processed by a computer for data analysis and report generation.

Core Component: Piezoelectric Ceramic Resonators

The sensitivity, stability, reproducibility, throughput and cost largely depend on the sensor component used. Piezoelectric ceramic resonators used should preferably possess high frequency and large surface areas, small size, and be compatible for immobilization of materials.

Ceramics are mass-sensitive transducers, their frequency being dependent on the mass of the crystal's surface as well as the mass of any layers confined to the electrode areas of the crystal. The technology of manufacturing stable, high frequency (500 kHz-1 GHz range) ceramic material is mature and these materials are readily available. Ceramic resonators with various frequencies (in the MHz range) and dimensions were tested first to choose the most sensitive and stable resonator, with a suitable surface coating for the immobilization of antibodies being used.

The trend for clinical diagnostic is moving towards the development of microarray format sample detection system that is suitable for high throughput screening. Thus miniaturization of the ceramic material is another issue that needs to be taken into consideration. The smallest dimension for commercial ceramic product is down to 0.4 mm×0.2 mm at a very low production cost compared to other piezoelectric materials like quartz. As the material can be produced in small size with stability, reproducibility and high sensitivity, it is very suitable for the development of a new generation of diagnostic kits with high throughput.

Selection of Markers: Cancer Markers as an Example

Cancer is the second leading cause of death worldwide and the total number of deaths due to cancers is 7.6 million accounting for 13% of total death worldwide. A WHO report in 2004 clearly indicated that lung cancer, stomach cancer, colon/rectum cancer, liver cancer, breast cancer and prostate cancer are the six most common types of cancer accounting for 60% of the total deaths caused by cancer. Thus, the subject cancer diagnostic biosensor will initially, focus on the simultaneous screening of a panel of cancer markers related to these six types of cancers.

Cancer markers are soluble molecules (usually glycoproteins) in the blood/urine that can be detected by monoclonal antibodies. Each cancer marker has a variable profile of usefulness for screening, diagnosis and prognosis, assessing response to therapy, and monitoring for cancer recurrence.

(1) Diagnostic functions of cancer markers: Screening tests require high sensitivity to detect early-stage disease. These tests also must have sufficient specificity to protect patients with false-positive results from unwarranted diagnostic evaluations. Biosensors for cancer markers offer significant benefits for the general population. In addition, cancer markers can play a crucial role in detecting disease and assessing response to therapy in selected groups of patients. Table 1 lists a number of common cancer markers used in clinical settings.

TABLE 1 Conditions Associated with Elevated Cancer Marker Levels Cancer Normal Additional associated Threshold marker value Primary tumor(s) malignancies Level Sensitivity CA 27.29 <38 units Breast cancer Colon, gastric, hepatic, >100 units Elevated in about 33% of per mL lung, pancreatic, per mL early-stage breast cancers ovarian, and prostate and about 67% of late-stage cancers breast cancers CEA <2.5 ng Colorectal cancer Breast, lung, gastric, >10 ng Elevated in less than 25% of per mL in pancreatic, bladder, per mL early-stage colon cancers nonsmokers medullary thyroid, head and 75% of late-stage colon <5 ng and neck, cervical, and cancers per mL in hepatic cancers, smokers lymphoma, melanoma CA 19-9 <37 units Pancreatic cancer, Colon, esophageal, and >1,000 units Elevated in 80% to 90% of per mL biliary tract hepatic cancers per mL pancreatic cancers and 60% cancers to 70% of biliary tract cancers AFP <5.4 ng Hepatocellular Gastric, biliary, and >500 ng Elevated in 80% of per mL carcinoma, pancreatic cancers per mL hepatocellular nonseminomatous carcinomasNonseminomato germ cell tumors us germ cell tumors: see b-hCG below b-hCG <5 mIU Germ cell tumors, Rarely, gastrointestinal >30 mIU AFP or b-hCG elevated in per mL gestational cancers per mL 85% of nonseminomatous trophoblastic germ cell tumors; elevated disease in only 20% of early-stage nonseminomatous germ cell tumors CA 125 <35 units Ovarian cancer Endometrial, fallopian >200 units Elevated in about 85% of per mL tube, breast, lung, per mL ovarian cancers; elevated in esophageal, gastric, only 50% of early-stage hepatic, and pancreatic ovarian cancers cancers PSA <4 ng Prostate cancer Undetectable level after >10 ng Elevated in more than 75 per mL for radical prostatectomy per mL percent of organ-confined screening prostate cancers NSE <4 ng Lung cancer, Also detected in patients >10 ng Elevated in more than 70 per mL neuroblastoma with Wilms' tumor; per mL percent of small cell lung melanoma; and cancers cancer. Measurement of of the thyroid, kidney, NSE level can provide testicle, and pancreas. information on the disease stage and the patient's prognosis and response to treatment. CA = cancer antigen; CEA = carcinoembryonic antigen; AFP = alpha-fetoprotein; b-hCG = beta subunit of human chorionic gonadotropin; PSA = prostate-specific antigen. Adopted from: G. L. Perkins, E. D. Slater, G. K. Sanders, and J. G. Prichard, “Serum Tumour Markers”, American Family Physician, 2003, Vol. 68, pp 1075-1082.

(2) Reasons for Targeted Cancer Markers

Cancer Antigen 27.29 (CA27.29): Cancer antigen (CA) 27.29 is a monoclonal antibody to a glycoprotein (MUC1) that is present on the apical surface of normal epithelial cells. CA 27.29 is highly associated with breast cancer, although levels are elevated in several other malignancies. CA 27.29 can also be found in patients with benign disorders of the breast, liver, and kidney, and in patients with ovarian cysts. However, CA 27.29 levels higher than 100 units per mL are rare in benign conditions.

Because of superior sensitivity and specificity, CA 27.29 has supplanted CA 15-3 as the preferred tumor marker in breast cancer. The CA 27.29 level is elevated in approximately one third of women with early-stage breast cancer (stage I or II) and in two thirds of women with late-stage disease (stage III or IV).

Carcinoembryonic Antigen (CEA): Carcinoembryonic antigen (CEA), an oncofetal glycoprotein, is overexpressed in adenocarcinoma, especially colorectal cancer. CEA elevations also occur with other malignancies. Non-neoplastic conditions associated with elevated CEA levels include cigarette smoking, peptic ulcer disease, inflammatory bowel disease, pancreatitis, hypothyroidism, biliary obstruction, and cirrhosis. Levels exceeding 10 ng per mL are rarely due to benign disease.

CEA values are elevated in approximately 50 percent of patients with tumor extension to lymph nodes and 75 percent of patients with distant metastasis. The highest values (above 100 ng per mL) occur with metastasis. The American Society of Clinical Oncology recommends monitoring CEA levels every two to three months for at least two years in patients with stage II or III disease who are surgical candidates.

Cancer Antigen 19-9 (CA 19-9): Elevated levels of CA 19-9, an intracellular adhesion molecule, occur primarily in patients with pancreatic and biliary tract cancers but also have been reported in patients with other malignancies. This tumor marker has a sensitivity and specificity of 80 to 90 percent for pancreatic cancer and a sensitivity of 60 to 70 percent for biliary tract cancer. T he positive predictive value of levels over 1,000 units per mL is 97 percent when CA 19-9 testing is used in clinical situations that are consistent with pancreatic cancer. Furthermore, CA 19-9 levels above 1,000 units per mL predict the presence of metastatic disease.

Alpha-Fetoprotein (AFP): Alpha-fetoprotein (AFP) is the major protein of fetal serum but falls to an undetectable level after birth. The primary malignancies associated with AFP elevations are hepatocellular carcinoma and nonseminomatous germ cell tumors. Patients with cirrhosis or viral hepatitis may have abnormal AFP values, although usually less than 500 ng per mL. Pregnancy also is associated with elevated AFP levels, particularly if the pregnancy is complicated by a spinal cord defect or other abnormality.

AFP levels are abnormal in 80 percent of patients with hepatocellular carcinoma and exceed 1,000 ng per mL in 40 percent of patients with this cancer. Retrospective studies showed improved survival with AFP screening. In patients with a hepatic mass and risk factors for hepatocellular carcinoma, an AFP level above 500 ng per mL is often used in lieu of biopsy to diagnose hepatocellular carcinoma.

Beta Subunit of Human Chorionic Gonadotropin (β-HCG): The β-hCG normally is produced by the placenta. Elevated β-hCG levels most commonly are associated with pregnancy, germ cell tumors, and gestational trophoblastic disease. Both AFP and β-hCG play crucial roles in the management of patients with nonseminomatous germ cell tumors. The AFP or β-hCG level is elevated in 85 percent of patients with these tumors.

Following AFP and β-hCG levels is imperative in monitoring response to treatment in patients who have nonseminomatous germ cell tumors. Patients with AFP and β-hCG levels that do not decline as expected after treatment have a significantly worse prognosis, and changes in therapy should be considered. Because curative salvage therapy is possible, the tumor markers are followed every one to two months for one year after treatment, then quarterly for one year, and less frequently thereafter.

Cancer Antigen 125 (CA 125): CA 125 is a glycoprotein normally expressed in coelomic epithelium during fetal development. Elevated CA 125 values most often are associated with epithelial ovarian cancer, although levels also can be increased in other malignancies. CA 125 levels are elevated in about 85 percent of women with ovarian cancer. Annual ultrasound examinations and CA 125 screenings have been advocated for women. Currently, ovarian cancer is treated with maximal surgical reduction, which leaves minimal clinical or radiographic disease. After definitive treatment of ovarian cancer, CA 125 levels should be obtained every three months for two years, and with decreasing frequency thereafter. Elevated CA 125 levels during follow-up nearly always indicate ovarian cancer recurrence.

Prostate-Specific Antigen (PSA): PSA is a glycoprotein produced by prostatic epithelium. The PSA level can be elevated in prostate cancer, prostatitis, benign prostatic hypertrophy, and prostatic trauma. Prostate cancer screening is suggested for black men at age of 40 years and in all men with a family history of prostate cancer. In patients without established risk factors, screening could begin at age 50. If elevated PSA values are confirmed, patients should be referred for biopsy. After treatment of prostate cancer, PSA levels should be obtained every six months for five years, and then annually. In men who have undergone radical prostatectomy, any detectable PSA is significant.

Neuron-specific enolase (NSE): NSE is the neurol isoenzyme of the intracytoplasmic enzyme enolase, which was first found in the extract of brain tissue and was later shown to be present in neuroendocrine cells and tumors. N CE is a well known marker of lung cancer. Previous studies show that increased values of NSE were present in cancer patients and that NSE was significantly correlated with tumour diameter and disease extent. Elevated serum concentrations of NSE have been found in over 70% of patients with small-cell lung cancer (SCLC). Measurement of NSE level in patients with lung cancer can provide information about the extent of the disease and the patient's prognosis, as well as about the patient's response to treatment.

The above eight protein cancer markers were chosen: CA25.29, AFP, CA 125, CA 19-9, CEA, β-HCG, NSE, and PSA. These markers cover the six types of most common cancers (lung, stomach, colon/rectum, liver, breast and prostate cancer) that account for more than 60% of the total cancer related death, and also relate to five other cancers types: neuroendocrine cancer, pancreatic cancer, digestive system organ cancer, ovary cancer and testicular cancer.

Each of the above markers are currently used either in isolation or in combination for a variety of cancers for screening, diagnosis, planning appropriate therapy, monitoring response to therapy (chemotherapy or radiotherapy), following-up care to check for recurrence or as a screening tool of those people in high-risk groups. A diagnostic technology that can detect the levels of all the above markers at the same time should offer the doctors a powerful tool in screening, diagnosis, and management of the most common cancers.

Immobilization of Antibodies

When the markers are selected e.g. cancer markers, their respective antibodies are immobilized on the ceramic material for detection of the antigen.

FIG. 4 is a schematic representation of the immobilization and antibody-antigen interaction in which the following are shown: a sample antigen 60, an antibody 62, a chemical layer 64, an electrode 66, a ceramic substrate 67 and an electrode 68.

Referring now to FIG. 4, in the ceramic sensor, sample antigen 60 is reacted with antibody 62 that is immobilized to electrode 66 through chemical layer 64. Ceramic substrate 67 is sandwiched between electrode 66 and electrode 68 and is connected to an oscillator for measuring of resonance frequency change induced by antigen-antibody interaction.

Piezoelectric ceramic resonators consist of ceramic wafers of different thicknesses sandwiched between two electrodes, which provide a means of connecting the device to an external oscillator circuit that drives the ceramic crystal to oscillate at its resonant frequency. For detection to take place, the antibody has to be immobilized on the ceramic surface first. There are many different methods for combining the ceramic sensor component with the biological sensing element (antibody against cancer marker), depending on the surface chemistry and the materials and the packing of the ceramic resonators. As an example, assuming that the antibodies will be immobilized on the electrode surface (which presumably is the most sensitive areas of the resonator), different types of antibody immobilization methods were tested to determine the more suitable method for antibody immobilization and sample detection.

(1) Self-Assembly Technique: Sulfo-LC-SPDP Chemistry

In one embodiment, an antibody is immobilized on the gold electrode's surface of a ceramic crystal by a self-assembled technique using sulfosuccinimidyl 6-[3′-(2-pyridyldithio) propionamido]hexanoate (sulfo-LC-SPDP). The highly ordered self-assembled monolayers (SAM) ensure well-controlled surface structure and offer many advantages to the performance of the sensor. The formation of SAM is due to the strong and spontaneous adsorption between sulfur atoms and a gold surface. Sulfo-LC-SPDP is a good thiol-cleavable heterobifunctional cross-linker, for it contains one N-hydroxysuccinimide residue and one pyridyl disulfide residue. The pyridyl disulfide residue of Sulfo-LC-SPDP can be used to introduce sulfhydryl groups into proteins or to conjugate an amine-containing protein to a sulfhydryl-containing protein such as IgG type of antibody.

The resulting disulfide bonds are cleavable by a reducing agent such as dithiothreitol (DTT). Thereafter, the thiolated antibody-gold complex is covalently formed on the gold electrode of the crystal.

The immobilization procedures are as follows:

-   -   The final concentration of an antibody for immobilization is         determined and optimized. The monoclonal antibody is diluted         with PBS (pH 7.4) to a suitable final concentration. Preferably,         a concentration of 0.05 to 2.5 mg/ml is used.     -   Equal volume of monoclonal antibody and cross-linker solution         (20 mM sulfo-LC-SPDP (water solution)) is mixed and incubated at         room temperature (RT) for 1.5 h.     -   To reduce the disulfide bond of the thiolated antibody, 2 μl of         DTT (0.1M sodium acetate buffer, 0.1M NaCl, pH 4.5) is added and         reacted for 30 min.     -   The mixture solution is then spread on the entire surface of one         gold electrode and dried for about 1 h at ambient temperature.         Preferred volume is from 0.2 to 5 μl.     -   The mixture solution is then spread on the entire surface of one         gold electrode and dried for about 1 h at ambient temperature.     -   The antibody-coated resonator, thus prepared was consecutively         washed with PBS and distilled water, then blown dry with a         stream of nitrogen gas.

The important advantages of using SAM as a platform for linking antibody molecules are easy formations of ordered, pinhole-free and stable monolayers, flexibility to design the head group of SAM with various functional groups in order to accomplish hydrophobic or hydrophilic surface as per the requirement, minimum amount of biomolecule needing for immobilization on SAM, and reasonable stability for extended period allowing several reliable measurements.

(2) Immobilization via Protein A (PA) Method

In another embodiment, protein A is used as the substrate for antibody immobilization. This is a commonly used two step immobilization process using protein A (PA) as the linker for antibody immobilization. PA can be spontaneously adsorbed onto the surface of the gold electrodes by physical adsorption to form the PA-gold complex. The affinity constant is 108 L/mol and the adsorption is nonspecific but compact. On the other hand, PA can covalently be combined with the Fc segment of IgG. The PA method's feature is that the immobilized antibody's functional fragment (Fab) is outwards of the crystal, which is advantageous to capture the antigen in sample solution.

The immobilization procedures are as follows:

-   -   Optimized amount of protein A solution in PBS (pH 7.2) is         dispersed on the entire surface of one gold electrode covering         the gold electrode area only. A preferred temperature and         incubation time is 4° C. for 16 h and RT for 2 h. Preferred         concentration of protein A is from 0.1 to 2 mg/ml and preferred         volume is from 0.2 to 5 μl.     -   After drying at RT, the resonator is rinsed with distilled water         to remove excessive PA solution.     -   Antibody at suitable concentration is applied to the electrode         surface, dried at RT, and washed subsequently with PBS and         distilled water. Preferred concentration of antibody is from         0.05 to 2.5 mg/ml.     -   After washing, the ceramic crystal is blown dry with a stream of         nitrogen gas.

(3) Immobilization via Sulfo-LC-SPDP-PA Method

In another embodiment, sulfo-LC-SPDP-PA is used as the substrate for antibody immobilization. This method combines the advantage of both the SAM and protein A.

The immobilization procedures are as follows:

-   -   The final concentration of protein A for immobilization is         determined and optimized. Protein A is diluted with PBS (pH 7.4)         to a suitable final concentration. Preferably, a concentration         of 0.1 to 2 mg/ml is used.     -   Equal volume of protein A and cross-linker solution (20 mM         sulfo-LC-SPDP (water solution)) is mixed and incubated at room         temperature (RT) for 1.5 h.     -   To reduce the disulfide bond of the thiolated antibody, 2 μl of         DTT (0.1 M sodium acetate buffer, 0.1M NaCl, pH 4.5) is added         and reacted for 30 min.     -   The mixture solution is then spread on the entire surface of one         gold electrode and dried for about 1 h at ambient temperature.         Preferred volume is from 0.2 to 5 μl.     -   The protein A coated resonator, thus prepared was consecutively         washed with PBS and distilled water, then blown dry with a         stream of nitrogen gas.     -   Antibody at suitable concentration was applied to the electrode         surface, dried at RT, and washed subsequently with PBS and         distilled water. Preferred concentration of antibody is from         0.05 to 2.5 mg/ml and preferred volume is from 0.2 to 5 μl.     -   After washing, the ceramic crystal is blown dry with a stream of         nitrogen gas.

(4) Nitrocellulose Coated Interface for Antibody Immobilization

In another embodiment, nitrocellulose is used as the substrate for antibody immobilization. Nitrocellulose is frequently used as a supporting surface for antibody immobilization and has wide application.

The immobilization procedures are as follows:

-   -   Place suitable size of nitrocellulose (NC) membrane on the         ceramic resonator gold centre.     -   Dissolve the NC membrane with 100% DMSO and allow it to dry at         room temperature. The preferred concentration of NC in DMSO is 1         mg/μl.     -   After drying at RT, a suitable amount of antibody is added onto         the NC coated on the gold centre. The preferred concentration of         antibody is from 0.05 to 2.5 mg/ml. and preferred volume is from         0.2 to 5 μl.

(5) Nafion Modified Interface for Antibody Immobilization

In another embodiment, nafion is used as the substrate for antibody immobilization. Nafion is a polyanionic perfluorosulfonated ionomer that is chemically inert, nonelectroactive and hydrophilic that can act as a matrix support with fast and simple immobilization procedure. The excellent thermal stability, chemical inertness and mechanical strength improves the operational and storage stability of the immunosensors. Proteins have an amphiphilic nature, and are adsorbed to the solid surface through the control of pH.

The immobilization procedures are as follows:

-   -   Dilute the 5% Nafion coating stock solution with buffer and         spread suitable amount on the gold surface of ceramic resonator.         The preferred final concentration of nafion is from 0.1% to 1%         and the preferred volume is from 0.2 to 5 μl.     -   After drying at RT, a suitable amount of antibody is applied on         the Nafion-modified ceramic resonator for 30 min at RT. The         preferred concentration of antibody is from 0.05-2.5 mg/ml and         the preferred volume is from 0.2 to 5 μl.     -   Different buffer and pH systems are tested for each marker to         find the suitable immobilization condition. The preferred pH for         the buffer is from pH 4.5-8.5, the buffer could be Phosphate,         Tris, Citrate, etc with a concentration of 10-50 mM.     -   Different NaCl concentration in the buffer was tested. The         preferred concentration of NaCl in the buffer is from 50-150 mM.     -   After incubation, the surface is rinsed with water, and is dried         at RT. Or     -   Dilute the 5% Nafion coating stock solution with PBS and mix         with suitable amount of antibody at 1:1 ratio. The preferred         final concentration of nafion is from 0.1% to 1%. The preferred         final concentration of antibody is from 0.05-2.5 mg/ml.     -   Spread a suitable amount of the mixture on the gold surface of         ceramic resonator and incubate at RT for 30 min. The preferred         volume is from 0.2 to 5 μl.     -   After incubation, the surface is rinsed PBS and equilibrated         with PBS for 200 min, and is dried at RT.

(6) Blocking and Binding Capacity

In another embodiment, a blocking reagent is applied before the detection was performed. A solution of 3% BSA in PBS is used to incubate with antibody-coated crystals to block the non-occupied residues to prevent non-specific interactions. The blocking procedures are as follows:

-   -   Dropping a droplet of BSA solution onto the coated surfaces for         1 h. The preferred volume is from 0.2 to 5 μl.     -   Washing with PBS and dried by a stream of nitrogen gas.

The frequency change of the ceramic resonators is measured after immobilization and blocking procedures respectively to ensure immobilization and blocking processes are successful. The frequency change ranging from 100 Hz to 50000 Hz depending on the type of chemistry is used. The binding capacity of the sensor can now be evaluated.

Individual Sensor Unit and Sensor Array

When the immobilization process is complete, the sensor detection unit for individual markers is assembled as shown in FIG. 5. FIG. 5 shows a single unit biosensor 52, a ceramic resonator 70, electrodes 72 and 74, a PCB 76 and an oscillator 78.

Referring to FIG. 5, s ingle unit biosensor 52 is shown having ceramic resonator 70 soldered on printed circuit board 76. Top electrode 72 on ceramic resonator 70 is immobilized with antibody 74 for antigen detection. The whole setup is connected to oscillator 78 that drives the ceramic resonator. Samples containing antigen are added to the device and the resonance frequency change is monitored using the oscillator and data acquisition. The decrease of frequency is monitored and compared to known standard concentrations of antigens to evaluate the amount of antigen in the sample.

Several issues need to be addressed:

(1). Sensor template: the sensor unit is soldered on a printed circuit board (PCB);

(2). Oscillating circuit: the circuits of various designs are tested to give the best performing stability;

(3). Liquid phase vs. gas phase: Liquid phase detection provides a real-time binding signal but may have the disadvantage of lower stability and sensitivity; Two-point (before and after) gas phase detection have proven sufficient to provide the needed information;

(4). Sample handling cells: whether the sample e.g. blood will directly contact the sensor unit or through strip by lateral flow technique where only the serum proteins are introduced to the sensor surface was tested.

When the individual sensor device is ready, an array of sensor units targeting at all the selected markers e.g. cancer markers are assembled on a printed circuit board for parallel screening of a panel of 16 markers simultaneously. The set up is shown in FIG. 6 that involves individual biosensor units 80 in an array format, a ceramic resonator 82, an electrode 84 and a PCB 86 are also shown.

As the dimension of ceramic resonators can be as small as 0.4 mm×0.2 mm, it is possible to assemble an array of 9-16 individual sensor units in an area of 1 cm². This offers another exciting possibility of developing a microplate (which is the standard testing format in clinical diagnosis where an array of 96 wells is manufactured on a plastic plate for parallel testing of dozens of samples) of sensor arrays. Such array of sensor arrays will have significant implications for most of the existing microplate-based in vitro diagnostic products in clinical applications.

Standard antigen detection was performed during each step of development to ensure that the sensor device is suitable for marker detection. The sample detection method and time is optimized during the testing procedure. When the microarray unit is ready, actual samples e.g. patient samples are used to evaluate the sensor performance.

Sample Introduction and Detection

In the preferred embodiment, several sample application methods are developed including sample addition using a lateral flow technique, direct sample application and sample addition using a microfluidic set up.

(1) Sample Introduction Using Lateral Flow Technique

In one embodiment, a sample addition strip for sample delivery using lateral flow technique is developed to press against the ceramic sensor surface; the site of interaction on which one antibody is immobilized and over which the sample e.g. serum passes during sample addition and lateral flow. FIG. 7 shows the design and set up of the sample application system: including a sensor 90 with two ceramic resonators 92, a connection to an oscillating circuit 94, a ceramic resonator 96 immobilized with a different antibody through different chemistry, a sample pad 98, an absorption pad 100, a sample solution 102, a sample 104 that moves laterally once loaded. This is followed by removing the sample loading cartridge and measuring the frequency at 106. As shown in FIG. 7, sensor 90 consists of two ceramic resonators 92 and is connected to oscillation circuit through connection 94. Ceramic resonators are immobilized with different antibodies through different chemistries as illustrated at 96. A sample loading cartridge consisting of sample pad 98 and absorption pad 100 is placed on top of the ceramic resonators that provide a carrier onto which a sample solution 102 is deposited. This sample then moves laterally as illustrated at 104 once deposited. The sample loading cartridge is removed and the resonance frequency is measured as illustrated at 106 after sample loading.

FIG. 8 shows the cartridge containing the sensor unit and lateral flow setup 110 showing the interior of the whole cartridge including the side view, sample loading cartridge 112 (Upper), a dual resonator chip 114 (Lower), an upper cover chip with label 116, a PCB 117, an absorption pad 118, a substrate pad 119 for pad assembly, a sample pad 120, a sample inlet 121, a cover chip with label 122, dual resonators 123, a PCB for resonator assembly 124, a bottom cover chip 125, and holes 126 for position alignment among different layers and connection to an oscillating circuit 127.

After coating the ceramic resonator gold centre with antibody, the biosensor is ready for sample analysis. A s shown in FIG. 8, sensors 123 are connected to PCB board 124 through standard soldering techniques (only two connection points are required for each resonator) and the whole PCB board is inserted into cartridge 112. Only the connection points to oscillating circuit 127 are exposed. The sample can be applied to the sensor by using the sample application system wherein this system consists of a cartridge containing a sample loading reservoir 121, the cover chip 122, the sample pad 120 and absorbent pad 118 that are assembled through substrate pad 119. This sample pad is placed against the surface of the ceramic resonator with immobilized antibody in the setup. There is only one sample inlet reservoir 121 which is located in the middle of the ceramic resonators as indicated in the figure. After sample application into the sample reservoir, the sample will flow due to capillary action laterally from the sample pad to the direction of the absorbent pad. An external pump is not required for sample application. There is no need for the preparation of special PMMA blocks that could generate a flow channel connecting the sensors either. When the sample passes through the ceramic resonator area with the immobilized capturing molecule, the antigen contained in the sample will interact specifically with the capturing molecule. Optimized time would be allowed for interaction between immobilized capturing molecule and antigen in the sample (the time would be no longer than 30 min). After the sample application, the PCB board containing the sensors is removed from the cartridge and the signal is ready for detection. This signal is compared to a pre-sample loading frequency and is correlated to the default standard to generate the result.

For an array type sensor, the reservoir is located in the central area as shown in the central square areas indicated by light color in FIG. 6. FIG. 9A shows the design and set up of the sample application system whereby a sample pad 132 and absorption pad 130 contained in the cartridge is placed on top of the microarray sensor units. The cartridge setup is similar to that shown in FIG. 8. When sample 134 is applied through the sample reservoir located in the middle, the sample containing the analyte flows laterally in the direction as indicated in the FIG. 9B towards absorption pad 136. When the sample passes through the ceramic resonator area with the immobilized capturing molecule, the antigen contained in the sample interacts specifically with the capturing molecule. Optimized time is allowed for interaction between immobilized capturing molecule and antigen in the sample (the time is no longer than 30 min). After the sample application, the PCB board containing the sensors is removed from the cartridge and the signal is ready for detection. This signal is compared to a pre-sample loading frequency and correlated to the default standard to generate the result. Again, an external pump is not required for sample loading.

Mass changes due to the binding antigen leads to a ceramic resonance frequency change in the device. The resonance frequency change is recorded using the oscillator and is transformed and recorded through data acquisition. The data is processed and compared to control samples for data analysis and a report is generated through custom designed analysis software.

(2) Setup for Direct Sample Application

In another embodiment, the sample is administrated directly onto the sensors through the reservoir on a cartridge. The analytes contained in the sample would interact with the capturing molecule immobilized on the ceramic sensor. FIG. 10 shows the design and set up of the sample application system showing a front view 140, bottom view 142, a sample loading reservoir 143, an absorption pad 144, a connection with solution when a spigot 145 is opened 145, a PCB sealing material 146, a PCB 147, a connection to an oscillating circuit 148, a cartridge with absorption pad and a sensor assemble 150, a spigot 151 to be opened after sample incubation and a ceramic resonator 152.

After coating the ceramic resonator gold centre with an antibody, the sensor is ready for sample analysis. As shown in FIG. 10, sensors 152 are connected to the PCB board 147 through standard soldering techniques (only two connection points are required for each resonator) and the whole PCB board is inserted into the cartridge containing absorption pad 144 as indicated in the figure. The sample is applied to the sensor by using the sample application system through the sample loading reservoir. Sample loading reservoir 143 contains spigot 145. After sample introduction, the analyte contained in the sample interacts specifically with the capturing molecule immobilized on the sensor. Optimized time allows for interaction between the immobilized capturing molecule and the antigen in the sample (the time is no longer than 30 min). After sample application, the spigot on the sample reservoir is opened, whereby the sample solution is absorbed by the absorbent pad contained in the cartridge. A washing buffer is added through the reservoir to remove non-specific binding agents which is also absorbed by the absorbent pad and the sensor chip is ready for measurement after solution removal. This signal is compared to a pre-sample loading frequency and correlated to the default standard to generate the result. By using this method, a sample is easily removed to prevent contamination and the whole chip is disposable which is very convenient in clinical applications.

For an array type sensor, the reservoir is located on the four sides of the microarray sensor chip as indicated in FIG. 11. FIG. 11 shows a bottom view 160, a front view 161, a PCB containing ceramic resonator 162, a sample reservoir with spigot 163, an absorption pad 164, a PCB assembly 165 with ceramic resonators and with sample loading setup, a connection to oscillation circuit 166, a cartridge with absorption pad 167 and resonators assembled, a sample loading unit 168 and a sample reservoir 169.

This figure shows the design and set up of the sample application system whereby the whole PCB containing the ceramic sensor array 162 is inserted into the cartridge containing absorption pad 164 as indicated. Sample 168 is applied to reservoir 163 at the dome-shaped middle point of the sensor chip. The applied sample flows to the sample reservoir and the analyte contained in the sample can specifically interact with the capturing molecule immobilized on the ceramic sensor. Optimized time is allowed for interaction between the immobilized capturing molecule and the antigen in the sample (the time is no longer than 30 min). After sample application, the spigot on the sample reservoir is opened, whereby the sample solution is absorbed by the absorbent pad contained in the cartridge. Washing buffer is also added at the middle of the sensor chip to remove non-specific binding agents which are also absorbed by the absorbent pad and the sensor chip is ready for measurement after solution removal. This signal is compared to a pre-sample loading frequency and correlated to the default standard to generate the result. By using this method, a sample is easily removed to prevent contamination and the whole chip is disposable which is very convenient in clinical applications.

(3) Sample Introduction Using Microfluidic Sample Application Setup

In another embodiment, the sample is administrated using a microfluidic sample application set up. The microfluidic-biosensor technology provides a unique opportunity to develop fractionation and a diagnostic platform for online separation of the sample e.g. blood cells, and detection of plasma markers e.g. cancer markers.

The microfluidic-sensor includes a separation unit of a microfluidic circuit for separating blood cells from plasma containing biomarkers. A sensing interface is provided comprising the specific antibody reacted against the selected biomarker (the antigen), and a transducer (such as ceramic resonator) is provided that converts the antibody-antigen binding event into an electrical signal as illustrated in FIG. 12. FIG. 12 shows a sample solution of whole blood 170, a microfluidic circuit 172, a ceramic sensor microarray zone 174, a single unit 175 of the ceramic sensor, an array type ceramic sensing unit 176 and signal detection and analysis 178.

In operation, and referring now to FIG. 12, sample solution 170 is applied to microfluidic circuit 172 where sample separation is carried out. The separated sample passes through the ceramic sensor microarray zone 174 that consists of array type ceramic sensing unit 176 which is made up of a single unit 175 of the ceramic sensor. The sample interacts with a capturing molecule immobilized on the ceramic sensors and the signals detected are transferred to a computation system for signal detection and analysis as shown at 178.

The sample e.g. a whole blood sample containing the analytes is fractionated by the microfluidic circuit to remove blood cells and allow plasma containing analytes to get into contact with the sensor for analysis. The specific antibodies (the recognition elements) against the selected markers e.g. cancer markers are immobilized on piezoelectric ceramic resonators for detection of plasma markers (antigens or analytes) presented in patient samples. Signals generated by the ceramic resonator including the oscillating frequency are modulated when the resonator-antibody interface undergoes certain changes due to the binding of the antigens to the immobilized antibodies.

Fabrication of Microfluidic Circuit

Rapid microdevice fabrication is demonstrated by molding PDMS against a PCB master. Channel system designs are generated in a CAD program (CorelDRAW 12.0, Corel Corporation, UK). Improved microfluidic structure fabrication technology is used for microfluidic structure fabrication.

2400 dpi transparencies are produced by a commercial printer from the CAD files serving as photomasks and are placed on top of a printed circuit board (PCB, Kinsten glassepoxy single sided, Chiefskill, Taiwan). Subsequently, the PCB is exposed in a standard PCB exposure unit (KVB-30 exposure unit, Chiefskill) for 80 seconds. Exposed PCB is then incubated in a developing agent (Chiefskill) for 10 minutes, rinsed and wet etched with ferric chloride for 1 hour. After etching, the PCB is thoroughly rinsed before acetone removal of the remaining photoresist.

The PCB master featured with microchannels is then covered with degassed PDMS prepolymer (10 base: 1 curing agent, Sylgard 184, Dow Corning, Midland, Mich.) which is followed by incubation at 65° C. for 1 hour. The cured PDMS replica is peeled off, trimmed and oxidized in a plasma cleaner (PDC-3XG, Harrick Scientific, Ossining, N.Y.) for 2 min together with another thin slab PDMS placed on a piece of cleansed glass slide. The replica is then sealed against the PDMS slab to form a microdevice for sample separation in the detection platform.

Blood Sample Fractionation

The plasma separation zone is a diffusion extraction device comprising microchannels with the shape illustrated in FIG. 13. This figure shows a microfluidic circuit 172, a ceramic sensor microarray zone 174, a blood inlet 180, a buffer inlet 181, a solution outlet 182, a plasma separation zone 183, a cell 184, a plasma containing biomarkers 185 and cells and waste 186. Buffer solution (75 μl) and a drop of sample e.g. whole blood (50 μl) is loaded into corresponding vials 181 and 180 sequentially. Whole blood and buffer solution are delivered into the microfluidic circuit by capillary action. A mixture of cells suspended in the whole blood sample stream enters the plasma separation zone 183 from the top while the buffer stream (extraction stream) enters from the bottom in the fashion illustrated in FIG. 13. These two streams dividing the microchannel (of plasma separation zone) in two equal halves because of their equal flow rate confined by the design. Due to the small size of the channels and the weak suction force employed for solution delivery, the flow is laminar and the streams do not mix. Under this laminar flow condition, molecular transport between blood and buffer streams occurs only by diffusion at plasma separation zone 183. Markers having a greater diffusion coefficient (smaller particles such as protein, sugars and small ions) have time to diffuse into the buffer stream 185 being extracted, while the larger biological cells 184 and waste 186 remain in the blood sample stream and are carried away towards solution outlet vial 182. In this way, blood cells are separated from the interested markers, and only makers are carried in the buffer stream and delivered towards the ceramic sensing units for detection. Whether or not in a single or microarray sensor format, only one sample is adopted, whereby the sample flows through all the sensing resonators in the flow pathway. T here is no need for external pumping or a controller system for the delivery. As will be appreciated, the signals are detected by a single integrated oscillating circuit for the array resonators, with one of the resonators used as a reference for control purposes.

Detection and Data Analysis

In one embodiment, the sensor in the system is made to oscillate. Upon the application of electricity, the resonance frequency is recorded, converted by an algorithm and is exported to a computer for data analysis. The pre-sample loading resonance frequency is recorded. The frequency after sample loading is compared to the pre-sample loading frequency. The frequency difference between pre- and post-sample application is analyzed by the computation system and correlated to the default standard based on relevant clinical data

Clinical Evaluation of the Sensors

In another embodiment, the working condition of the sensor is optimized using standard antigen samples. Clinical samples (serum) from both normal and diseased cohorts are tested and compared to results obtained from standard ELISA assays and clinical diagnosis. Further improvement on the sensor can be done based on the clinical feedback.

Other Sensors Developed Based on the Ceramic Sensor Platform

The sensor technology platform developed is easily used for the development of other sensors with different applications e.g. in vitro diagnostic tests.

The worldwide market size of the total in vitro diagnostic testing market is forecast to grow to US$45.5 billion by 2010, with point-of-care diagnostic tests accounting for ⅓^(rd) of the total worldwide in vitro diagnostic testing market. In one embodiment, the subject sensor is used for diagnosis of heart disease.

Cardiovascular Disease (CVD):

CVD is the leading cause of death worldwide causing 16,733,000 deaths that account for 29.3% of the death rate. Heart disease is the second leading cause of death in Japan (15.5%), Hong Kong (14.6%) and the fourth leading cause of death in China (14.4%). Around 80% of all CVD deaths worldwide took place in developing, low and middle-income countries, while these countries also accounted for 86% of the global CVD disease burden. It is estimated that by 2010, CVD will be the leading cause of death in developing countries. As well, at least 20 million people survive heart attacks and strokes every year, a significant proportion of them requiring costly clinical care, which puts a huge burden on long-term care resources. The economic cost of cardiovascular diseases and stroke in 2002 is estimated at US$329.2 billion. The true cost in human terms of suffering and lost lives is incalculable.

Cardiovascular diseases impose a heavy socioeconomic burden on different countries. In the five largest European countries, France, Germany, Italy, Spain, and the United Kingdom, a total of US$110 to US$115 billion is spent annually for the management of cardiovascular disorders. Of this total, approximately US$40 billion is spent for treating coronary heart disease, more than US$15 billion for stroke, US$12 billion for hypertensive disease, and approximately US$8 billion for congestive heart failure.

Cardiovascular is the leading therapeutic category with global sales of US$50 billion a year. With cardiovascular disease as the leading cause of morbidity and mortality worldwide, the challenge now is to develop increasingly sensitive diagnostic modalities, especially in vitro diagnostic tests that can solve the problem of early diagnosis or detection of predisposing factors. Cardiovascular diagnostic product sales, including electrocardiography, noninvasive imaging, and intravascular techniques, generated more than US$1.4 billion in 1999 for the five major countries of Europe. Key drivers are forecasted to boost the market for cardiac diagnostics from US$14 billion in 2001 to US$22 billion in 2006, 12% per year. The development is emphasized on in vitro diagnostic tests, primarily because this is where the major market opportunities lie.

There are a number of well known cardiac markers that are easily incorporated into the subject sensor platform to develop fast screening sensors for early detection of different heart diseases.

In another embodiment, sensors for autoimmune diseases and coagulation disorders detection also have large market potential.

Autoimmune Diseases:

Autoimmune diseases comprise 80 or more chronic disabling diseases that affect almost every organ system in the body e.g. nervous, gastrointestinal, endocrine system, skin, skeletal and vascular tissues. In each disease, the immune system may produce autoantibodies to the endogenous antigens, with consequent injury to the host tissues and organs. In many diseases the presence of autoantibodies antedates the disease itself.

It is estimated that the total European autoimmune disease diagnostics market will reach US$700 million in 2011 and the global autoimmune diagnostic market is worth US$2 billion. The growth is mainly due to two reasons 1) Identification of many diseases that are of autoimmune disorders in which the disease pathogenesis was not known previously e.g. Celiac disease; 2) Availability of therapeutics for many autoimmune disorders increase the need for autoimmune diagnostics for early identification and disease control.

The presences of the autoantibodies in these diseases can be detected through the subject sensor platform for fast screening, early detection and disease monitoring and control for different autoimmune diseases.

Coagulation Disorders:

Coagulation disorders (coagulopathies) are disruptions in the body's ability to control blood clotting, an essential function of the body designed to prevent blood loss. Coagulation, or clotting, is a complex process (called the coagulation cascade) that involves 12 coagulation factors found in blood plasma and several other blood components. Each factor has a precise role in coagulation. Besides the factors, plasma carries a number of other proteins that regulate bleeding e.g. platelets. A deficiency in clotting factors or a disorder that affects platelet production or one of the many steps in the entire process can disrupt clotting and severely complicate blood loss from injury, childbirth, surgery, and specific diseases or conditions in which bleeding can occur. Coagulation disorders arise from different causes and involve different complications.

Coagulation disorders affect millions of lives worldwide. Thus, every hospital needs to undertake coagulation testing resulting in more than 25,000 clinical laboratories performing coagulation testing globally. The worldwide coagulation testing market is valued at US$925 million and is growing at 10% per annum. Prothrombin time tests alone account for US$125 million.

The application of the subject sensor platforms for simultaneous detection of various coagulation disorders will enhance the efficiency of the disease screening process.

In another embodiment, the subject sensors are used in applications including the detection of food contaminants and monitoring of pollutants.

Food Safety:

The subject platform technology is used for developing sensor products for application in areas other than bio-medical application e.g. food safety. With increasing environmental pollution and awareness of the health, consumers are demanding safer and healthier food.

Food analysis has a global market of US$1.4 billion which is shared by about 50 companies whereas USA and Europe companies make up ⅔ of them. Food microbiological tests have a global market of US$705 million in which rapid testing methods account for US$145 million or 20% of the total market share.

The subject sensor can be used for food analysis in food industry to monitor food manufacturing process to ensure food safety during production. The sensor could also be used by the end user at home to monitor food safety for both fresh and processed food e.g. the subject sensor may be used for monitoring of multiple food pathogens: Clostridium botulinum; Salmonella typhi; Salmonella paratyphi; Shigella dysenteriae; Vibrio cholerae; E. coli; Listeria monocytogenes, etc.

While the present invention has been described in connection with the preferred embodiments of the various figures, it is to be understood that other similar embodiments may be used or modifications or additions may be made to the described embodiment for performing the same function of the present invention without deviating therefrom. Therefore, the present invention should not be limited to any single embodiment, but rather construed in breadth and scope in accordance with the recitation of the appended claims 

1. A sensor for detection of a marker in a sample comprising: a piezoelectric resonator sensor operating in the thickness extensional mode and having a frequency range of 500 kHz-1 GHz; a source for applying an oscillating electric field across said sensor; a sample loading cartridge; a sample at said sensor; a circuit for measuring the resonant frequency change of said sensor as a result of said sample being placed at said sensor, and, A computational system communicably connected to the said sensor, said computational system gives reading on the marker amount that correlates to the normal marker amount in said sample.
 2. The sensor of claim 1, wherein said piezoelectric sensor operates in the thickness extensional mode.
 3. The sensor of claim 1, wherein said sensor includes array of said piezoelectric resonators, said resonators aligned and separated by a gap, one of said resonators being a reference resonator and the other of said resonators having a coating layer on an electrode surface thereof adapted to capture a molecule in said sample for simultaneous detection of multiple markers.
 4. The sensor of claim 1, wherein said ceramic resonator is a high frequency synthetic polycrystalline ferroelectric ceramic includes lead titanate zirconate with gold electrode and operates in thickness extension mode.
 5. The sensor of claim 1, wherein said sensor has a surface coated with capture molecules for markers.
 6. The sensor of claim 5, and further including a recorder for recording the binding interaction between the capture molecule and the marker, and wherein said measuring circuit includes a processor for correlating the measured marker frequency differential to the default standard concentration in said sample.
 7. The sensor of claim 1, wherein said samples are taken from the group consisting of blood, serum, plasma, urine non-organic solutions and gas.
 8. The sensor of claim 1, wherein said samples are used for the group of analyses consisting of disease diagnosis, food analysis, environmental pollution analysis and biodefense.
 9. The sensor of claim 5, and further including a sample loading cartridge and a computation system for monitoring marker levels in said samples, wherein the sample loading cartridge consists of a sample introduction setup for the group of setups consisting of a direct sample application setup, a sample addition strip and a microfluidic sample introduction setup.
 10. The sensor of claim 1, wherein said sample has molecules captured by capturing molecules on a surface of said sensor and wherein the binding interaction between a capturing molecule and a marker is measured in terms of resonance frequency shift.
 11. The sensor of claim 5, wherein said circuit measures the resonance frequency change before and after sample application, said resonance frequency change correlated to the mass of the marker being captured by the capture molecule on said ceramic resonator surface.
 12. The sensor of claim 11, wherein said source applies an oscillating electric field across said sensor and wherein said circuit measures at least one resonance frequency of said sensor as the pre-sample application resonance frequency and wherein, after providing the sample and incubating for interaction of the capture molecule and the marker and after applying said oscillating electric field across said sensor, at least one resonance frequency of the sensor is measured as the post-sample resonance frequency, whereby said circuit correlates the frequency change before and after sample application to said sensor to surface mass and/or surface density, whereby the surface mass and/or surface density is correlated with the amount of marker bound by the capture molecule.
 13. The sensor of claim 1, wherein the mass change represented by resonant frequency is correlated to a default standard mass range.
 14. The sensor of claim 1, wherein said sensor is mounted on a printed circuit board and wherein said frequency source includes an oscillator. 